Stacking gel necessary? - (Sep/02/2008 )
I am supposed to be making a 20% polyacrylamide gel for the purpose of separating short dsRNA, but am not sure if I need to make my gel with a stacking gel or not??
Does anyone know if and why the stacking gel is necessary?? If so what percentage should it be at?
Also, when I run the dsRNA on the 20% gel, it frowns...does this have anything to do with the absence of the stacking gel?
If I'm not wrong the stacking gel is for proteins. For nucleotides I had used 8-15% acryl-bis with no stacking gel.
The frowning is cause by temperature, so 2 soln. are: 1. use less voltage or 2. run the gel at 4C.
I never use a stacking gel for RNA gels. To stop the frowning I put just loading buffer in 2 or 3 wells either side of the sample wells and run the gel at constant watts (15W for medium sized gels) - this helps keep the temperature of the gel relatively constant.
Does anybody know why a stacking gel is used for proteins and not nucleic acids???
The stacking gel help to "pack" the proteins so they will enter all at the same time into the resolving gel.
So why is this not necessary for nucleic acids??
nucleotides have a negative charge, peptides have positive, negative and neutral so the SDS-PAGE neutralize the electrical charge by the SDS detergent and change of pH between the stacking and resolving gels. The stacking made a peptide "pack" so all of them will enter at the same time to the resolving gel and it will separate only by weight not by electrical charge. If not using the stacking there is a possibility that you will have peptides that will never leave the wells.